The existing PCR methods using commercially available equipment are generally based on utilization of polymer tubes that are installed in metallic heating blocks. The high thermal mass of standard heating blocks with tubes installed in them and samples, the low thermal conductivity of the walls of the tubes restrict heating and cooling rates of the sample and may lead to high temperature non-uniformity across the sample in the tube.
When using usual laboratory devices intended for PCR in polymer tubes or plates the recommended time of maintaining the temperature during thermal cycling is 2 minutes or more. Ausubel et al., eds. (1996) Current Protocols in Molecular Biology, Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., for example, recommend using a five-minute duration per cycle excluding time for temperature transitions. As a result, the PCR analysis consisting of 40 thermal cycles would take 3 or more hours to be completed using typical PCR equipment with 20-50 μL sample volume.
In order to decrease the time spent on one cycle there have been recently proposed many methods of PCR analysis in miniature reaction containers. To increase the time required for temperature transition for the mass of the heating element, the mass of the containers and the volume of the samples in such devices have been considerably decreased, which allowed reduction of the thermal mass, use of materials with high thermal conductivity coefficients and a higher ratio of the sample surface area to its volume. Methods of contactless heat supply to the sample are also used for heating. For example, RapidCycler (Idaho Technologies Inc., USA) allows a relatively fast change of the PCR mixture temperature during temperature transition and ensures a relatively effective heat transfer from the heater to the samples. In this device 30 cycles of rt-PCR may be completed within 10 minutes.
There are also methods implemented in microfluidic devices that reduce the time required for one cycle. Kopp et al. (1998) Science, 280:1046, for example, describes a device where the PCR mixture successively flows through a microchannel in the form of a meander in the microfluidic chip through three temperature zones, which results in cyclic change of the PCR temperature during 20 thermal cycles. As the cross-section of the microchannel is relatively small, the temperature of the solution inside the microchannel is set rather fast. The time during which the mixture is at a certain temperature is regulated in this case by the flow rate. The authors demonstrated the possibility of PCR in the described device with the cycle time of 6.6 seconds.
Thus, application of microchip technologies allows significant decrease in the PCR cycling time, which leads to decrease of the total PCR analysis time and increased throughput.
However, rt-PCR implementation in such microfluidic devices faces with certain difficulties.
The higher ratio of the surface area between the microreactor and the sample to the volume leads to decrease of polymerase activity and even to irreversible inactivation of the enzyme. The surfaces of such materials as silicon, metals, quartz, and glass demonstrate irreversible sorption of DNA and enzyme, as well as other components of the reaction. To eliminate the restrictions it is necessary to add substances that prevent sorption and deactivation of PCR components, such as amino acids, peptides and surfactants, into reaction mixture [U.S. Pat. No. 6,660,517 “Mesoscale polynucleotide amplification devices”]. The same patent also displays some methods that allow PCR microreactor surface passivation. Such protection layers prevent adsorption and inhibition of PCR components, which allows for the achievement of high sensitivity of PCR analysis.
One of the problems known to specialists in PCR analysis is evaporation of the sample during the thermocycling phase. Since temperature of DNA denaturation is close to the boiling point of water, intensive evaporation of PCR mixture during the reaction can inhibit the PCR flow, which is generally eliminated in standard devices either by insulating the mixture water surface from the atmosphere by means of a liquid immiscible with water, by mineral oil, for example, or by using a heated lid to seal the reaction vessels.
In closed channels of microfluidic chips the microreactors can be sealed with valves in the microchannel [U.S. Pat. No. 7,118,910 “Microfluidic device and methods of using the same”]. Implementation of this method of sealing is technically complicated and increases the cost of the analysis using such a microchip.
Prevention of evaporation during thermocycling for microchips containing open-well microreactors is usually achieved by insulation of the reaction mixture from the atmosphere by application of a liquid immiscible with water on the surface of the aqueous solution of the reaction mixture, mineral or silicone oil, for example [U.S. Pat. No. 6,664,044 “Method for conducting PCR protected from evaporation”]. However, in the described device an ink-jet system is repeatedly used to inject the working PCR and samples, which may lead to uncontrolled degradation of PCR reagents and internal contamination of the samples in the process of application. Another method and device [USA patent application 20070196237 “Apparatus for regulating the temperature of a biological and/or chemical sample and method of using the same”] uses a microchip with reaction volumes on the substrate surface that are a droplet of a liquid immiscible with water, with the PCR mixture placed in it. However, in this method the reaction mixture is separated from the heated substrate surface by a layer of the liquid immiscible with water, which slows down the heating and cooling process. Moreover, the reaction volumes in this method can uncontrollably move, since the entire surface of the heat-conducting substrate is hydrophobic. It may lead to spatial mismatch of the heated zone and the heated sample in the process of the sample introduction and thermal cycling, which will result in unreliable results of the PCR analysis.
Another shortcoming common for the known microfluidic devices is the increased labour intensity in mixing the samples with the PCR mixture components prior to introduction into the microchip. Besides, this procedure requires costs for additional consumables (plastic tips, tubes) and is practically unrealizable in usual tubes with the volume of the handled liquids of less than one microliter due to the increased probability of evaporation of the reagents and samples during mixing. There are methods that allow immobilization of one or several components of the reaction mixture necessary for the PCR in the microreactor to fill the microreactor with a aqueous solution containing nucleic acids and the remaining components of the PCR mixture [U.S. Pat. No. 7,118,910 “Microfluidic device and methods of using the same”]. However, this method is labour-intensive and is hard to control during production as it requires application of biological reagents directly in the process of the microchip manufacture, which increases the danger of the negative impact of subsequent technological processes on the applied reactants (impact of increased temperature, chemical compounds, emission).
There are methods that involve lyophilization of the prepared PCR mixture containing all or almost all PCR reagents and additional stabilizers in the tubes [RF patent 2259401 “Dry mixture of reagents for polymerase chain reaction and method of PCR analysis”, U.S. Pat. No. 6,153,412 “Lyophilized reagent for polymerase chain reaction”]. This method can be implemented practically in any microreactor system with open reactors.
Despite the large number of available methods and devices of rt-PCR implementation in microfluidic and microchip formats there is still a high need for development of new, improved methods and devices. This technical field has a need for a cost-effective method of express quantitative identification of nucleic acids of a variety of samples having high sensitivity without large labour costs in preparation for the analysis as well as the need for a device for its implementation.
The closest analogue to an exemplary embodiment of the present application may be said to be the method and the device described in the USA patent application 20070196237 “Apparatus for regulating the temperature of a biological and/or chemical sample and method of using the same”. The application discloses the method of conducting biochemical reactions, including rt-PCR, containing stages of thermocycling and fluorescent signal detection using a thermoregulation module comprising a heater and temperature detector. A substrate of a heat-conducting material is placed on the module and a biological sample is applied on the substrate, for example, a mixture containing all components for real-time PCR analysis that is insulated from the atmosphere by introducing into a virtual reaction cell formed by a liquid immiscible with water, by mineral oil, for example.
The main drawbacks of the prototype are the low real heating and cooling rates of the sample; technological complexity of the thermoregulation module with integrated microheaters and thermosensors, which results in higher costs and reduces the commercial attractiveness of the device. Another drawback of the analogue is the uncontrolled sensitivity reduction at the possible contact of the sample with the surface of the heat-conducting material. In addition, the method and device disclosed in the analogue description require preliminary preparation of the solutions of PCR components, mixing of PCR components with the sample and introduction of the resulting mixture into reaction zones, which leads to additional labour costs, higher risk of operator's errors and higher costs of analysis due to the growing quantity of consumables (tubes, tips for the dosing unit, reactants). Another drawback of the analogue is the possibility of spatial mismatch of the heated zone and the heated sample in the process of the sample introduction and thermal cycling, which will result in unreliable results of the PCR analysis.